Molecules that bind to and inactivate the HIV-1 nucleocapsid protein (NCp7) are currently being evaluated as new antiviral drugs. In particular, derivatives based on a 2-mercaptobenzamide thioester template (SAMTs) have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7 via acyl transfer from the thioester to the sulfur of a zinc-coordinating cysteine residue. These zinc-binding domains are excellent targets for the development of new antiretroviral and microbicidal agents because of their structural conservation and the broad range of function of NCp7 in the viral life cycle. Our efforts have focused on the evaluation of the therapeutic potential of the SAMT compounds and the identification of new analogs with increased activity. Our previous work identified three SAMT compounds that were shown to be virucidal and to inhibit cell-to-cell associated transmission of HIV-1 in co-culture systems. In addition, based on mutational analysis of the NCp7 amino acid sequence, we were able to extend the previously reported mechanism of action of the SAMT compounds to include a secondary S to N intramolecular acyl transfer that occurs after the primary acyl transfer from the thioester to a cysteine side chain in the protein. Based upon this detailed mechanism of action of SAMT compounds, we have synthesized novel derivatives of the SAMT chemotype that are predicted to be more stable than the SAMT compounds. This improvement may enable the new compounds to be used systemically to treat HIV. We have studied the in vitro metal ejection activity of a series of these compounds and found that some have similar activity as the lead SAMT compounds. In addition, preliminary mass spectrometry experiments demonstrate that the new compounds also follow a similar reaction mechanism as the SAMTs. The new compounds are currently being tested for antiviral activity in co-culture systems. We have begun to explore the use of the SAMT compounds as potential microbicide candidates. We found that three lead SAMT compounds prevent cell-to-cell transmission of HIV and inhibit the dissemination of virus from cervical explants. We further determined that the SAMTs retain their activity in gel formulation and in synthetic mucus. Furthermore, they did not induce vaginal irritation in rabbits. A preliminary study found that a SAMT lead prevented infection in five of six rhesus macaques challenged in a vaginal dual-infection trial. These experiments were encouraging, and we have continued to pursue the use of the SAMTs as topical microbicides to prevent the transmission of HIV. Recent studies have found that gel-based microbicides are hampered by patient compliance, suggesting that this method of delivery would not be optimal for a good microbicide. Thus, we initiated a collaboration with Dr. Karl Malcolm at the Queens University Belfast in Ireland to study the release of the SAMT compounds from silicone rings, such as those currently approved for use as birth control. We have determined that certain lead SAMTs are able to be sufficiently released from the ring in vitro . We have also begun a collaboration with Dr. James Smith at the Centers for Disease Control and Prevention to study the inhibition of virus transmission in non-human primates using the vaginal rings. Preliminary results have established that the SAMT in the ring is released in vivo similarly to what was measured in vitro . Studies are currently underway to assess the efficacy of a SAMT compound in a ring to block viral transmission. Finally, we are using mass spectrometry experiments to elucidate the metabolism and pharmacokinetics of the SAMT compounds. In collaboration with the Waters Corporation, we have identified a panel of potential metabolites of the SAMT compounds. We are working to validate the chemical structure of the metabolites and evaluate the possibility that some may have antiviral activity. In addition, we are preparing to identify metabolites of the SAMT compounds from biopsy tissue. We are continuing to investigate the activity and metabolism of the lead SAMT compounds in cellular model systems and non-human primates. Identification of a potentially safe and efficacious single or combination candidate microbicide in non-human primates, and the elucidation of their pharmacokinetics, will lead us to more formal testing in preparation for clinical evaluation.